A collaboration of two labs at the Morehouse School of Medicine (Drs. Bond and Powell) have been investigating the possibility that soluble Nef, secreted from HIV infected cells, is responsible for a significant portion of the T-cell depletion that is a hallmark of HIV pathogenesis. Our research group has developed a body of results that support a key role for Nef in T-cell depletion. We have been funded to do a pilot study to begin to examine this overall theory in Rhesus macaques. However, there remain a number of key issues revolving around the mechanics of how secreted Nef induces the apoptosis of T-cells leading to pathogenesis. The focus of this proposal is on two key aspects of Nef-induced T-cell depletion and is addressed in this proposal through the hypothesis that Nef protein is secreted from HIV infected cells in vesicular form and induces an apoptotic signal in non-infected T-cells through key interactions between the Nef apoptotic motif (M1) and the amino terminal domain of CXCR4. Questions regarding the secretion of Nef from HIV-1 infected cells (Dr. Powell's lab will be lead group), will be addressed in the following AIMS: AIM 1: Demonstrate that Nef is secreted from transfected and infected T-cells and characterize the secreted vesicular bodies;AIM 2: Determine if mutations outside of the Nef apoptotic region influence induction of apoptosis or incorporation of Nef into vesicles. Questions regarding the detailed interactions between Nef and the CXCR4 receptor (Dr. Bond's lab will be lead group) will be addressed in the following AIMS: AIM 3: Determine those amino acids on the Nef apoptotic motif 1 and on CXCR4 critical to Nef-binding and apoptosis;AIM 4: Determine whether other nonstandard CXCR4 ligands follow the same rules of interaction. Together these results should provide a more complete picture of the mechanics of secreted Nef induction of apoptosis in T-cells leading to the progression toward AIDS.